State of the Marine Environment along the Bulgarian Coast as Indicated by Acetylcholinesterase Activity of Donax trunculus L. Almira P. Georgieva, Elina R. Tsvetanova, Nesho H. Chipev, V. Alexandrova Institute of Neurobiology, Bulgarian Academy of Sciences, 23, Acad. G. Bonchev, str., 1113 ,

BACKGROUND RESULTS

Anthropogenic pressures are increasingly affecting marine ecosystems, causing The activities of AChE in the soft tissue of D. trunculus gathered in March and in significant changes in their state and functionality. The Black Sea is a unique semi- June were significantly (p<0.05) lower than the activities in the samples from July and еnclosed basin which is accepted to be one of the highly polluted seas. As the number September (Fig. 2). of possible pollutants is huge they can have not only cumulative but also synergistic In March the AChE activities, measured in the samples from and effects. Thereby assessment of the biological responses of susceptible organisms, along Slanchev Bryag were significantly lower than that from . In June, the with the qualitative and quantitative analysis of chemical contaminants, is becoming a lowest AChE activities were observed in wedge clams sampled from Slanchev Bryag key tool in monitoring programs. Currently, a number of biomarkers, as well as their and Arkutino The activity of the enzyme in the July samples from Pasha Dere, Rodni combinations, are used to assess the impacts of pollution, environmental risk and Balkani and Azalia was almost twice as high as those from June (Fig. 2). The AChE marine environmental management. Two types of biomarkers are conventionally activity in the samples of clams, gathered in September varied from about 30 to 48 distinguished - those that reflect the exposure of organisms to environmental stressors U/mg protein. The lowest average values were measured in the samples from (Exposure Biomarkers) and those that show the adverse effect of stressors on the state Arkutino and (Fig. 2). The highest activity of AChE was detected in the of organisms (Effect Biomarkers). Inhibition of acetylcholine esterase (AChE) is used samples from , gathered in September. as a marker of the exposure and effect mainly of organophosphorus pesticides, carbamate, heavy metals, polycyclic aromatic hydrocarbons (PAHs), polychlorinated 60 biphenyls (PCBs), dichlorodiphenyltrichloroethane (DDT), detergents, algal toxins, and recently microplastics in aquatic organisms. It is well established that inhibition 50 of AChE in marine organisms is mainly caused by neurotoxic effects . 40 AIM The aim of the present study was to make a preliminary assessment of the 30 ecological and ecotoxicological risks associated with the contamination of Black Sea

sublittoral sandy habitats using AChE in the wedge clam Donax trunculus L. as a bio- 20 U/mg protein U/mg indicator. 10

0

Duni(S)

Irakli(N)

Azalia(N)

Tsarevo(S)

Arkutino(S) Arkutino(S)

Primorsko(S) Primorsko(S)

Sveti Vlas(S) Sveti

Pasha Dere(N) Pasha

Rodni Balkani(N) Rodni

Chernomorets(S)

Slanchev bryag(S) Slanchev

Slanchev Bryag(S) Slanchev Sv.Kons&Helena(N) March June July September

Fig. 2. Acetyl choline esterase activity in soft tissue of D. trunculus gathered from MATERIALS AND METHODS different localities and seasons (S – Southern localities; N – Northern localities) Sampling. Donax trunculus Adult wedge clams L. (23-35 mm) were collected in The enzyme activity in the clams from Arkutino and Primorsko were significantly 2020 from 12 localities of their natural shallow sublittoral sandy habitats along the higher at higher temperature of the marine water - the average temperature for the Bulgarian Black Sea coast (Fig. 1) in three seasons: spring (March), summer (June-July) localities for the time of sampling in June was +20.8°C, and in September +27.4°C and autumn (September). In order to confirm the significance of the observed patterns in AChE activity in the wedge clams from the sampled localities in different seasons we applied ANOVA Locality N E test and the results are presented in Table 1. The main effect of “locality” alone was Azalia 43.2360 28.0184 Table 1. Results of ANOVA test of effects of highly significant and reflected the locality and season on AChE in D. trunculus Sv. Konstantin & significant variations observed in the AChE 43.2294 28.0146 MS activity in the wedge clams sampled from Effects df F p Effect the different localities (Table 1). Similarly, Rodni Balkani 43.2026 27.9243 the factor “season” had a significant Locality 7 572.82 13.43 0.00000 individual effect. The interdependent effect Pasha Dere 43.1212 27.9284 of ”locality” and “season” proved to be also Season 2 4028.19 94.47 0.00000 highly significant (Table 1). Hence the effects 42.7498 27.8901 of the state of the marine environment of a Locality* locality on AChE activity in the wedge clams 8 564.59 13.24 0.00000 Sveti Vlas 42.7091 27.7595 Season living there can be different depending on the season. Slanchev Bryag 42.6906 27.7137 The results obtained showed that the wedge clams from locations near to sources of Chernomorets 42.4468 27.6422 increased coastal anthropogenic impact, had lower AChE activity, as follows: - the sites Sveti Vlas and Slanchev Bryag, in March; 42.3662 27.6927 - the sites Arkutino, Slanchev Bryag, Dyuni and Primorsko, in June. - the sites Arkutino, Primorsko and resort Sv. Konstantin and Elena, in September Arkutino 42.3334 27.7312 Higher values of AChE were measured in samples from Irakli, whichc is an area falling within the protected site "Emine". Primorsko 42.2738 27.7565 In contrast, in clams from Tsarevo significantly higher AChE values were present – over 30% higher than those from Arkutino and Primorsko, and over 20% higher than Tsarevo 42.1744 27.8504 those from Sv. Konstantin and Elena resort. Although on average in the samples from the northern regions higher values of the Fig. 1. Sampling localities along the Bulgarian Black Sea coast with geographical co- AChE in the wedge clams were found, compared to those from the southern localities, ordinates of the sampling sites. the differences were rather individual and seemed to be due to the state of the marine environment of the location. Determination of acetylcholinesterase (AChE) activity. Acetylcholinesterase activity was assayed by the method of Ellman et al. (1961), and has been calculated CONCLUSION as U (μM ACh/min)/mg of protein. The presented results, although preliminary, strongly indicated that AChE was significantly reduced in wedge clams sampled from localities adjacent to areas known as Statistical analyses. All measurements are presented as mean ± standard error sources of relatively high anthropogenic load such as the bigger resorts, i.e. Slanchev (SE). Significance of differences between groups was estimated using t-statistic and Bryag, Sveti Vlas, Arkutino and Dyuni. Inhibition of AChE can reduce the capacity to ANOVA, at p < 0.05 significance level. The analyses were carried out using the burrow into the substrate, feeding rate and locomotion impairment and even mortality STATISTICA software package (STATSOFT Inc., Tulsa, USA). in calms probably corresponding to 70-90% inhibition.

Acknowledgements: This work was supported by grant КП-06-Н31/6 of National Science Fund, Bulgaria.